Metabolite from streptomyces strain NRRL accession No. B-30145 and mutants thereof for controlling plant diseases

ABSTRACT

A novel antibiotic-producing  Streptomyces  sp. is provided that exhibits antifungal activity only on certain specific plant pathogens. Also provided is a method of treating or protecting plants from fungal infections comprising applying an effective amount of an antibiotic-producing  Streptomyces  sp. having all the identifying characteristics of NRRL Accession number B-30145. The invention also relates to fungicidal compositions comprising this novel  Streptomyces  strain and the antibiotics and metabolites produced by this strain either alone, or in combination with other chemical and biological pesticides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.09/671,943 filed Sep. 27, 2000. now U.S. Pat. No. 6,524,577. Thecontents of this application are hereby incorporated by reference intothe present disclosure.

FIELD OF THE INVENTION

The present invention is in the field of biopesticides.

BACKGROUND OF THE INVENTION

For a number of years, it has been known that various microorganismsexhibit biological activity so as to be useful to control plantdiseases. Although progress has been made in the field of identifyingand developing biological pesticides for controlling various plantdiseases of agronomic and horticultural importance, most of thepesticides in use are still synthetic compounds. Many of these chemicalfungicides are classified as carcinogens by the EPA and are toxic towildlife and other non-target species. In addition, pathogens maydevelop resistance to chemical pesticides (Schwinn et al., 1991).

Biological control offers an attractive alternative to syntheticchemical fungicides. Biopesticides (living organisms and the compoundsnaturally produced by these organisms) can be safer, more biodegradable,and less expensive to develop.

The actinomycetes, including the streptomycetes, are known producers ofantifungal metabolites (Lechavalier and Waksman, 1962; Lechavalier,1988). Several actinomycete-produced antibiotics are routinely used inan agricultural setting such as streptomycin and terramycin for fireblight control.

Streptomycetes have demonstrated both in vitro and in vivo activityagainst plant pathogens. Axelrood et al. (1996) isolated 298actinomycetes from Douglas-fir roots. Approximately 30% of these strainsdemonstrated antifungal activity against Fusarium, Cylindrocarpon,and/or Pythium in vitro. Yuan and Crawford (1995) reported thatStreptomyces lydicus WYEC 108 showed both strong in vitro antifungalactivity and inhibition of Pythium root rot in pot tests with pea orcotton seed. Reddi and Rao (1971) controlled Pythium damping-off intomatoes and Fusarium wilt of cotton with Streptomyces ambofaciens.Rhizoctonia root rot was controlled by Streptomyces hygroscopicus var.geldanus (Rothrock and Gofflieb, 1984). These authors reported that thecontrol was dependent on the in situ geldanamycin concentration producedby this strain. The same authors also saw protection of soybeans fromPhytophthora megasperma var. sojae by Streptomyces herbaricolor andStreptomyces coeruleofuscus (1984). Chamberlain and Crawford (1999) sawin vitro and in vivo antagonism of turfgrass fungal pathogens by S.hygroscopicus strain YCED9. Crawford (1996) patented the use of thisstrain to control plant pathogens in U.S. Pat. No. 5,527,526. Suh (1998)patented 2 Streptomyces sp. that were active against Rhizoctonia solaniand Phytophthora capsici. A Streptomyces griseoviridis product againstFusarium spp. and other soil pathogens is on the market as Mycostop™.

SUMMARY OF THE INVENTION

A novel antibiotic-producing Streptomyces sp. is provided that exhibitsantifungal activity only on certain specific plant pathogens. Alsoprovided is a method of treating or protecting plants from fungalinfections comprising applying an effective amount of anantibiotic-producing Streptomyces sp. having all the identifyingcharacteristics of NRRL Accession number B-30145. The invention alsorelates to fungicidal compositions comprising this novel Streptomycesstrain and the antibiotics and metabolites produced by this straineither alone, or in combination with other chemical and biologicalpesticides.

The antibiotic-producing Streptomyces sp. can be provided as asuspension in a whole broth culture or as an antibiotic-containingsupernatant obtained from a whole broth culture of anantibiotic-producing Streptomyces sp. Also provided is a novelbutanol-soluble antibiotic that exhibits specific antifungal activityand a process for isolating the novel butanol-soluble antibiotic.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is the analytical HPLC chromatogram of active fraction 6.(Microsorb C18, 10 cm×4.6 mm, 100 Å, flow rate 1 mL/min, UV detection at220 nm, acetonitrile+0.05% TFA/water+0.05% gradient as follows: 0-30min, 5-65%; 30-40 min, 65-100%; 40-45 min, 100%).

FIG. 1B is the UV spectrum of the active peak eluting at 14.755 minutesin the chromatogram described in 1A.

FIG. 2A is the analytical HPLC chromatogram of active fraction 7 underthe same conditions described in 1A.

FIG. 2B is the UV spectrum of the active peak eluting at 16.146 minutesin the chromatogram described in 2A.

FIG. 3 is the C-8 HPLC chromatogram of the methanol eluate from theDiaion HP-20 resin step described in Method B. (HP Zorbax Eclips XDB-C8column, 5 μm, 150×4.6 mm, flow rate 0.8 mL min, UV detection at 220 nm,chart speed 2 mm/min. Solvent A, 25:5:70 acetonitrile/methanol/water.Solvent B, 65:5:30 acetonitrile/methanol/water. Gradient: 100% A at 0minutes increased to 3% B over 20 minutes.)

FIG. 4 is the ¹H NMR spectrum (400 MHz, CD₃OD) of the semi-pure activemetabolite obtained from purification method A.

FIG. 5 is the ¹³C NMR spectrum (100 MHz, CD₃OD) of the semi-pure activemetabolite obtained from purification method A.

FIG. 6 is the LC ESI-MS (Liquid Chromatography ElectroSpray Impact-MassSpectrum) of Peak A obtained from purification method B. (Microsorb C18, 10 cm×4.6 mm, 100 Å, flow rate 1 mL/min, acetonitrile+0.02%TFA/water+0.02% gradient as follows: 0-30 min, 5-65%; 30-40 min,65-100%; 40-45 min, 100%).

FIG. 7 is the LC ESI-MS (Liquid Chromatography ElectroSpray Impact-MassSpectrum) of Peak B obtained from purification method B. (Microsorb C18,10 cm×4.6 mm, 100 Å, flow rate 1 mL/min, acetonitrile+0.02%TFA/water+0.02% gradient as follows: 0-30 min, 5-65%; 30-40 min,65-100%; 40-45 min, 100%).

DETAILED DESCRIPTION

The present invention provides a novel strain of Streptomyces sp. ormutants thereof with antifungal activity only on specific plantpathogens such as Alternaria, Phytophthora, Botrytis, Rhizoctonia andSclerotinia. This novel strain was deposited with the NRRL on Jul. 20,1999 under the provisions of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purpose of PatentProcedure under Accession No. B-30145. The invention also includesmethods of preventing and treating fungal diseases in plants using suchbacterial strains or antibiotic-containing supernatants or pureantibiotics obtained from such bacterial strains. The invention alsoincludes a butanol soluble antifungal antibiotic with a molecular weightof less than 10,000 daltons, with stability to base and to heattreatment of 1 hour at 80° C. and lability to acid treatment.

Definitions

The singular form “a,” “an” and “the” include plural references unlessthe context clearly dictates otherwise. For example, the term “a cell”includes a plurality of cells, including mixtures thereof.

The term “comprising” is intended to mean that the compositions andmethods include the recited elements, but not excluding others.“Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andagriculturally acceptable carriers. “Consisting of” shall mean excludingmore than trace elements of other ingredients and substantial methodsteps for applying the compositions of this invention. Embodimentsdefined by each of these transition terms are within the scope of thisinvention.

As used herein, “biological control” is defined as control of a pathogenor insect by the use of a second organism. Known mechanisms ofbiological control include enteric bacteria that control root rot byout-competing fungi for space on the surface of the root. Bacterialtoxins, such as antibiotics, have been used to control pathogens. Thetoxin can be isolated and applied directly to the plant or the bacterialspecies may be administered so it produces the toxin in situ.

The term “fungus” or “fungi” includes a wide variety of nucleatedspore-bearing organisms that are devoid of chlorophyll. Examples offungi include yeasts, molds, mildews, rusts, and mushrooms.

The term “bacteria” includes any prokaryotic organism that does not havea distinct nucleus. “Pesticidal” means the ability of a substance toincrease mortality or inhibit the growth rate of plant pests.

“Fungicidal” means the ability of a substance to increase mortality orinhibit the growth rate of fungi.

“Antibiotic” includes any substance that is able to kill or inhibit amicroorganism. Antibiotics may be produced by a microorganism or by asynthetic process or semisynthetic process. The term, therefore,includes a substance that inhibits or kills fungi for example,cycloheximide or nystatin.

“Antifungal” includes any substance that is able to kill or inhibit thegrowth of fungi.

The term “culturing” refers to the propagation of organisms on or inmedia of various kinds. “Whole broth culture” refers to a liquid culturecontaining both cells and media. “Supernatant” refers to the liquidbroth remaining when cells grown in broth are removed by centrifugation,filtration, sedimentation, or other means well known in the art.

An “effective amount” is an amount sufficient to effect beneficial ordesired results. An effective amount can be administered in one or moreadministrations. In terms of treatment and protection, an “effectiveamount” is that amount sufficient to ameliorate, stabilize, reverse,slow or delay progression of the fungal or bacterial disease states.

“Positive control” means a compound known to have pesticidal activity.“Positive controls” include, but are not limited to commerciallyavailable chemical pesticides. The term “negative control” means acompound known not to have pesticidal activity. Examples of negativecontrols are water or ethyl acetate.

The term “solvent” includes any liquid that holds another substance insolution. “Solvent extractable” refers to any compound that dissolves ina solvent and which then may be isolated from the solvent. Examples ofsolvents include, but are not limited to, organic solvents like ethylacetate.

The term “metabolite” refers to any compound, substance or byproduct ofa fermentation of a microorganism that has pesticidal activity.Antibiotic as defined above is a metabolite specifically active againsta microorganism.

The term “mutant” refers to a variant of the parental strain as well asmethods for obtaining a mutant or variant in which the pesticidalactivity is greater than that expressed by the parental strain. The“parent strain” is defined herein as the original Streptomyces strainbefore mutagenesis. To obtain such mutants the parental strain may betreated with a chemical such as N-methyl-N′-nitro-N-nitrosoguanidine,ethylmethanesulfone, or by irradiation using gamma, x-ray, orUV-irradiation, or by other means well known to those practiced in theart.

A “composition” is intended to mean a combination of active agent andanother compound, carrier or composition, inert (for example, adetectable agent or label or liquid carrier) or active, such as anadjuvant. Examples of agricultural carriers are provided below.

We describe a novel antibiotic-producing strain of Streptomyces sp. NRRLNo. B-30145 and mutants thereof that have antifungal activity only onspecific plant pathogens. Also provided is a supernatant isolated fromthe culture as well as a composition comprising the culture. In afurther aspect, the compositions further comprise at least one chemicalor biological pesticide.

A metabolite produced by the Streptomyces sp. strain is also provided bythis invention. The metabolite exhibits activity against plantpathogenic fungi and is heat and base stable, is acid labile and has amolecular weight of less than 10,000 daltons. By way of example, themetabolite may have a molecular weight [M+H⁺] between about 925 tobetween about 865.

The one or more metabolites produced by the Streptomyces sp. strainexhibit UV absorption between about 215 nm and 220 nm. The metabolitemay be comprised of a variety of molecules including, but not limitedto, propargyl alcohol segments [C═C—CH(OH)], oxygenated methine carbons(X—CH—Y) or a sugar moiety. By way of example, the metabolite maycomprise at least two propargyl segments, several oxygenated methinecarbons (by way of example, e.g., 5 to 10) and/or a sugar moiety.Alternatively, the one or more metabolites produced by the Streptomycessp. strain may share the same carbon skeleton and differ in degree ofoxygenation.

The present invention also provides antifungal compositions comprising ametabolite produced by Streptomyces and isolated according to a methodcomprising:

(a) loading a whole broth culture of Streptomyces sp. strain NRRL No.B-30145 or mutants thereof having all the identifying characteristics ofNRRL No. B-30145 onto a non-ionic absorbent polymeric resin;

(b) eluting the metabolite with an alcohol;

(c) screening the eluate of step (b) with a bioassay for fractions ofthe eluate exhibiting antifungal activity;

(d) loading the fractions of the eluate exhibiting antifungal activityof step (c) on a HPLC column; and

(e) eluting the metabolite with an organic solvent.

The method may further comprise washing the resin with water prior tostep (b) and screening the eluate of step (e) with a bioassay to selectthe fractions exhibiting antifungal activity.

The whole broth culture of step (a) may be freeze dried and re-suspendedwith an aqueous solution (e.g., water) prior to adding to the non-ionicabsorbent polymeric resin. In a preferred embodiment the whole brothculture added to the resin is a homogenized cell-free whole brothculture. Examples of non-ionic absorbent polymeric resin that may beused include, but are not limited to, Supelco Sepabead SP-207 or SupelcoDiaon HP-20.

The eluent used to remove the metabolite in step (b) may be an alcoholor a gradient of aqueous alcohol. By way of example, methanol or agradient of aqueous methanol may be used as the eluent (e.g., Example6).

The bioassay of step (c) may be any assay which evaluates antifungalactivity. Examples of such bioassays include but are not limited to, theagar diffusion assay or slide germination assay. For example, thebioassay may be a germination assay with Monilinia fructicola and/orAlternaria brassicicola.

Examples of an HPLC column that may be used in step (d) include, but arenot limited to, C-18 or C-8. Examples of the organic solvent that may beused to remove the metabolite from the HPLC column include, but are notlimited to, an acetonitrile-water gradient (e.g., Example 6).

The metabolite can also be formulated as a composition, with a carrieror alternatively, with at least one chemical or biological pesticide.

In order to achieve good dispersion and adhesion of compositions withinthe present invention, it may be advantageous to formulate the wholebroth culture, supernatant and/or metabolite/antibiotic with componentsthat aid dispersion and adhesion. Suitable formulations will be known tothose skilled in the art (wettable powders, granules and the like, orcan be microencapsulated in a suitable medium and the like, liquids suchas aqueous flowables and aqueous suspensions, and emulsifiableconcentrates). Other suitable formulations will be known to thoseskilled in the art.

The strain, culture, supernatant and isolated metabolite are useful toprotect or treat plants, fruit, and roots from fungal infections byapplying an effective amount of the active formulation to the plant,fruit or root. The formulations are particularly suited to treat orprevent infections caused by a fungus selected from the group consistingof Alternaria solani, Botrytis cinerea, Rhizoctonia sp., Sclerotiniasp., and Phytophthora sp.

All patents and publications cited herein are incorporated by reference.Full bibliographic citations for these may be found at the end of thespecification, immediately preceding the claims.

The following examples are provided to illustrate the invention. Theseexamples are not to be construed as limiting.

EXAMPLES Example 1

Characterization of Strain NRRL No. B-30145

NRRL No. B-30145 was identified based on 16S rRNA sequencing. Theprotocol used to generate the 16S rRNA gene data sequence (AcculabCustomer Handbook v. 1.0) is described as follows.

The 16S rRNA gene is PCR amplified from genomic DNA isolated frombacterial colonies. Primers used for the amplification correspond to E.coli positions 005 and 531. Amplification products are purified fromexcess primers and dNTPs using Microcon 100 (Amicon) molecular weightcut-off membranes and checked for quality and quantity by running aportion of the products on an agarose gel.

Cycle sequencing of the 16S rRNA amplification products is carried outusing AmpliTaq FS DNA polymerase and dRhodamine dye terminators. Excessdye-labeled terminators were removed from the sequencing reactions usinga Sephadex G-50 spin column. The products are collected bycentrifugation, dried under vacuum and frozen at −20° C. until ready toload. Samples are re-suspended in a solution of formamide/bluedextran/EDTA and denatured prior to loading. The samples areelectrophoresed on an ABI Prism 377 DNA Sequencer. Data are analyzedusing PE/Applied Biosystem's DNA editing and assembly software. Onceobtained, sequences are compared against PE/Applied Biosystem'sMicroSeq™ database using MicroSeq™ sequence analysis software. Sequencesare also compared to the GenBank and Ribosomal Database Project (RDP).

The result of the 16S rRNA sequencing identified NRRL No. B-30145 as aStreptomyces sp. This strain may belong to the species S. mashuensis(formerly Streptoverticillium mashuense) or a related species, assuggested by the sequencing results. The best match was Streptomycesmashuensis with a 98% match score.

Example 2

Activity of NRRL No. B-30145 Against Plant Pathogens in In-Vitro Culture(Zone Assay).

NRRL No. B-30145 was tested against an array of different plantpathogens utilizing two different in-vitro assays. The agar diffusion(zone) assay consists of applying either plant pathogen spores over thesurface of an agar medium to create an entire lawn of growth orutilizing a mycelial agar plug placed in the center of the petri dishthat will grow and colonize the agar. Circular wells approximately 7.0mm in diameter are removed from the agar using a pipette attached to avacuum pump. Fermentation samples of NRRL No. B-30145 are added to eachwell along with known standards and water checks. Plates are incubatedfor three to four days under environmental conditions conducive for eachpathogen. Results consist of a zone of no pathogen growth around thewell or a greatly reduced amount of pathogen growth around the well orno affect. The size and type of zone is recorded for each sample.Results for NRRL No. B-30145 in agar diffusion assays are presented inTable 1. Results within agar diffusion were variable; diffusion throughagar may be inhibited.

TABLE 1 Activity of NRRL No. B-30145 against selected plant pathogens inthe agar diffusion (zone) assay. Alternaria brassicicola No Zone/WeakActivity Botrytis cinerea Weak Activity Monilinia fructicola No ZonePhytophthora capsici Moderate activity Pythium sp. Weak ActivityColletotrichum acutatum No Zone Rhizoctonia solani No Zone Sclerotiniasclerotiorum No Zone

The second type of in-vitro assay performed to test the pathogenspectrum of NRRL No. B-30145 was the slide germination assay.Fermentation samples of NRRL No. B-30145 at various dilutions were addedto glass depression slides (25 mm×75 mm with 18 mm diameter depression1.75 mm deep) and an equal volume of pathogen spores were mixed with thesample. Slides were incubated on moistened paper towels in sealedplastic boxes at room temperature overnight. Results are determined byobserving the fermentation sample/spore suspension sample using acompound microscope at 100×. Typical results consist of lack ofgermination of the pathogen propagule or greatly reduced germinationand/or growth. In addition, various types of malformations of theinitial growth from the pathogen spores may occur. The spectrum ofactivity of NRRL No. B-30145 is presented in Table 2. Completeinhibition of spore germination occurred at low concentrations offermentation samples.

TABLE 2 Activity of NRRL No. B-30145 against selected plant pathogens inthe slide germination assay. Alternaria brassicicola No GerminationAlternaria dauci No Germination Botrytis cinerea No GerminationMonilinia fructicola No Germination

Example 3

Activity of NRRL Against Plant Pathogens in Plant Bioassay Tests.

Activity of NRRL No. B-30145 was tested against tomato late blight(Phytophthora infestans), tomato early blight (Alternaria solani), graymold (Botrytis cinerea), turf brown patch (Rhizoctonia sp.), and peanutsouthern blight (Sclerotinia minor). All tests were conducted undercontrolled environment in the laboratory with plant material grown fromseed under typical commercial greenhouse conditions.

Tomato Late Blight—Phytophthora infestans

The pathogen is grown on rye agar in standard petri dishes at 16° C. inthe dark. Sporangia are collected by flooding the plate with water andscraping the mycelium to dislodge the sporangia. The sporangialsuspension is passed through cheesecloth, quantified and adjusted to1.0×10⁴. Tomato seedlings at the 3^(rd) to 5^(th) leaf stage are sprayedto run-off with the fermentation sample of NRRL No. B-30145 using anartist airbrush at 40 psi. Treated seedlings are allowed to air dry atroom temperature for at least two hours then inoculated with thesporangial suspension by lightly spraying the upper surfaces of thetomato seedlings using a hand held sprayer. Inoculated seedlings areplaced in solid bottom flats filled with water and then are covered witha plastic dome to maintain leaf wetness. Flats are incubated at 20° C.with a 14-hr photoperiod for four days continuously covered by theplastic domes. Seedlings are then rated based on a disease rating scalefrom 0-5 with 0 equaling no symptoms, and 5 equaling 75% or more of thefoliage colonized by the pathogen. A typical example of a late blighttest is presented in Table 3.

TABLE 3 Results of NRRL No. B-30145 treated tomato seedlings against thelate blight pathogen Phytophthora infestans. Treatment Ave. D.I.Replications 1-4 Sample 990702 1.1 1.0 0.5 2.0 1.0 Sample 990709 1.1 1.02.0 1.0 0.5 Sample 990825 1.3 1.5 1.0 1.5 1.0 Sample 990913 1.0 1.0 1.01.5 0.5 Quadris 30 ppm 0.1 0 0.5 0 0 Water Check 4.3 4.0 4.0 5.0 4.0Samples are different fermentations of NRRL No. B-30145.D.I. is Disease Index.Tomato Early Blight—Alternaria solani

The pathogen is first grown on commercial Difco potato dextrose agar(PDA) at 22-25° C. under 14-hour lights until the entire plate iscovered. The fungus and the agar medium is then cut into small squaresapproximately 10 mm square and placed fungus side up on a specializedsporulation medium (S-Medium: 20 g sucrose, 20 g calcium carbonate, 20 gBacto-agar per liter). The S-Media plates are flooded with a thin layerof water and incubated 2-3 days at 22-25° C. under 14-hour lights untilfull sporulation of the pathogen occurs. Plates are then flooded withwater and the agar squares are scraped from the plate. The suspension ispassed through cheesecloth and the spores are quantified and adjusted to1.0×10⁵. Tomato seedlings at the 3^(rd) to 5^(th) leaf stage are thensprayed until run-off using an artist airbrush as described previously.Treated seedlings are allowed to dry and then inoculated with the sporesuspension. Seedlings are placed in flats and covered as describedpreviously and incubated at 25° C. with a 14-hour photoperiod. Seedlingsare rated based on a scale of 0-5 as previously described. Results froma typical test are presented in Table 4.

TABLE 4 Activity of NRRL No. B-30145 against the early blight pathogenAlternaria solani. Treatments Ave. D.I. Replications Test-1 Sample990216 1.0 2.0 0.5 0.5 Quadris 60 ppm 1.8 1.5 2.5 1.5 Water Check 4.03.0 4.0 5.0 Test-2 Sample 990216 1.1 1.5 1.0 1.0 1.0 Water Check 4.5 5.04.0 4.0 5.0D.I. is Disease Index.Pepper Gray Mold—Botrytis cinerea

The pathogen is grown on standard PDA under a 14-hour photoperiod at 22°C. until the fungal growth has completely covered the plate (7-9 days).Spores are collected by flooding the plate with water and then gentlyscraping with a spatula to dislodge the spores. The spore suspension ispassed through cheesecloth and quantified and adjusted to 1.5×10⁶.Pepper seedlings are grown until the 4^(th) to 6^(th) true leaf stageand fermentation samples are sprayed on the upper leaf surfaces using anartist airbrush as described previously. Treated seedlings areinoculated, placed in flats and covered with plastic domes. Flats areplaced at 20° C. under continuous darkness for 2.5 days. Seedlings arerated on a 0-5 scale as described previously. Table 5 depicts resultsfrom two typical tests.

TABLE 5 Activity of NRRL No. B-30145 against Botrytis cinerea TreatmentAve. D.I. Replications Test-1 Sample 990216 1.4 1.5 1.5 1.5 1.0 Break 20ppm 0.1 0 0 0.5 0 Water Check 4.0 4.0 4.0 3.0 5.0 Test-2 Sample 9902161.9 1.5 2.0 2.0 2.0 Break 20 ppm 0.8 0 1.5 1.0 0.5 Water Check 4.5 4.05.0 5.0 4.0 D.I. is Disease Index.Turf Brown Patch—Rhizoctonia sp.

Two ml of fermentation sample was added to each cell of a 6-cell pot ofone-month old turf seedlings (Bentgrass). A 4 mm mycelial plug of a 2-3day old culture of Rhizoctonia sp. was placed under the soil surface.Each treatment was replicated 6 times. Inoculted pots were placed inplastic flats and covered with a plastic dome. The flats were placed ona light rack (16 Hr/day) and incubated at room temperature. Diseaseseverity was evaluated after 5-6 days incubation and compared with thewater treated control. The results indicated that NRRL No. B-30145 has asuppressive activity against Rhizoctonia (Table 6).

TABLE 6 The efficacy of NRRL No. B-30145 on turf disease caused byRhizoctinia sp. Dilution Treatment Factor Rep 1 Rep 2 Rep 3 Rep 4 Rep 5Rep 6 30145 1x +* + + + + + 30145 ½x + + + + + + Water +++ +++ +++ ++++++ +++ *“+” = light symptoms, “+++” = severe symptomsPeanut Southern Blight—Sclerotinia minor

Peanut seedlings at the first 2-leaf stage were treated with NRRL No.B-30145 and a 4-mm mycelial plug is placed on the base of each stemafter the treated plants dried. Inoculated plants were incubated in adew chamber for 2 days before being placed in a plastic flat sealed witha cover dome. The flat was incubated on a light rack (16Hr/day) at roomtemperature for 10 days. Disease severity was assessed by comparing thetreated with the water control. The results (Table 7) indicated NRRL No.B-30145 whole broth at 1× has some controlling activity againstSclerotinia minor.

TABLE 7 The efficacy of NRRL No. B-30145 on peanut Sclerotinia blight.Treatment Dilution Factor Rep 1 Rep 2 Rep 3 30145 1x +/− 0 +/− 30145 ½x++ ++ + 30145 ¼x 0 ++ ++ Water +++ +++ +++ *“+/−” indicates strongsuppression, 0 indicates no infection, “+” = light symptoms, “+++” =severe symptoms,.

Example 5

Antifungal Metabolite Produced by NRRL No. B-30145.

The whole broth of NRRL No. B30145 was partitioned into ethyl acetate,butanol and aqueous fractions. Each fraction was tested againstAlternaria brassicicola in a spore germination assay. The Alternariaspores were germinated in the presence of each sample in depressionmicroscope slides containing 40 μl of sample and 20 μl of pathogenspores. Approximately 16 hours later the spores are observed under amicroscope to see if they have germinated. No germination (score of 0)compared to the water control (100% germination and growth=score of 5)indicates activity of the sample being tested. Results of the Alternariagermination assay with different NRRL No. B-30145 fractions are shownbelow (score on a 0 to 5 rating as above).

Score Fraction Rep 1 Rep 2 Rep 3 Ethyl acetate 3 nd 4 n-butanol 0 0.2 1Aqueous 0 5 5 Whole broth 0 0.2 0 Water Check 5 5 5

The metabolite is clearly in the butanol soluble fraction and is notreadily extractable in ethyl acetate. Other characteristics of themetabolite were determined. The molecule was shown to pass through a10,000 molecular weight cut off filter indicating the metabolite issmaller than 10,000 daltons. The activity was not lost when treated withbase or upon heating to 80 degrees C. for one hour. The activity waslost when treated with acid (the score against Alternaria increased from0 to 5).

Fractionation of the butanol extract on octadecylsilane bonded to silicagel (ODS) flash chromatography using an acetonitrile (ACN)/watergradient with 0.01% trifluoroacetic acid (TFA) yielded an activefraction eluting with 50% acetonitrile/water with 0.01% TFA. Fractionswere tested in an Alternaria germination assay for activity (0-5 ratingscale).

Fraction Score ODS 10% ACN 4 ODS 20% ACN 5 ODS 50% ACN 0.5 ODS 100% ACN5 Water Check 5

Further purification by ODS HPLC yielded 2 active components (Fraction 6and 7) from an isocratic elution with 31% acetonitrile in water with0.02% TFA).

Fraction Score HPLC Fr. 1 5 HPLC Fr. 2 5 HPLC Fr. 3-5 4 HPLC Fr. 6 3HPLC Fr. 7 2 HPLC Fr. 8 5 HPLC Fr. 9 5 Water Check 5

The HPLC chromatogram of the active 50% acetonitrile/water with 0.01%TFA flash chromatography fraction and the HPLC chromatograms of theactive fractions 6 and 7, including UV spectra of the active principles,are shown in FIGS. 1 and 2.

NRRL No. B-30145 most closely matched Streptomyces mashuensis by 16S RNAsequencing. Unlike the antibacterial metabolites typically associatedwith S. mashuensis, the fungicidal activity of NRRL No. B-30145 wasextractable with butanol. S. mashuensis is known to producestreptomycin, which is a water-soluble antibacterial compound. Anotherantibiotic produced by S. mashuensis, monazomycin (Akasaki et al. 1963),does not display a shoulder at 215-220 nm as does the fungicidal activefractions of NRRL No. B-30145.

Antifungal compounds have also been found in the closely related andpossibly synonymous species Streptomyces griseocarneum (American TypeCulture Collection). These include porfiromycin (Claridge et al., 1986),a purple compound whose corresponding UV spectrum is not seen in theactive fraction of NRRL No. B-30145 and the Heptaenes trichomycin(Komori and Morimoto, 1989) and griseocarnin (Campos et al., 1974),whose corresponding UV spectra are also not present in the activefraction. The fungicidal active is also not neutramycin, which isextractable with ethyl acetate (Mitscher and Kunstmann, 1969).

Example 6

Additional Methods for Further Purification of the Antifungal Metaboliteof NRRL No. B-30145

Method A

The freeze-dried whole broth culture was re-suspended in water (2.0 L)and loaded onto a column containing a non-ionic polymeric resin (SupelcoSepabead SP-207; 26×3.0 cm) equilibrated in water. The column was washedwith water (200 mL) and then with a gradient of aqueous methanol asfollows: (1) 20:80 methanol/water (200 mL), (2) 40:60 methanol/water(200 mL), (3) 60:40 methanol/water (200 mL), 80/20 methanol/water (200mL), and (5) methanol (200 mL).

Bioassay results (germination assay with Monilinia fructicola and/orAlternaria brassicicola) indicated that all fractions were active. Eachfraction was individually fractionated on octadecylsilane-bonded silicagel (ODS) HPLC using an acetonitrile/methanol/water (TOSOHASS ODS-80TS;10 μm, 21.5×30 cm. Solvent system: solvent A:acetonitrile/methanol/water 25:5:65, solvent B:acetonitrile/methanol/water 65:5:30. Gradient: start at 0 min withsolvent A and hold for 25 min. Then increase solvent B to 35% over 50min. Flow=6.0 mL/min). All fractions yielded approximately the same HPLCprofile with the activity located at two regions: peak A (t˜55-63 min)and peak B (t˜65-70 min). Peak B was further fractionated on anotherreversed-phase HPLC column (Phenomenex Luna Phenyl-Hexyl; 5 μm, 250×10mm. Solvent system: solvent A: acetonitrile/methanol/water 25:5:65,solvent B: acetonitrile/methanol/water 65:5:30. Gradient: start at 0 minwith solvent A and hold for 15 min. Then increase solvent B to 25% over25 min. Flow=2.0 mL/min). One major component was isolated; however,analytical HPLC analysis indicated a high-UV absorbing contaminant thatco-eluted with active metabolite. Therefore, an alternative purificationmethod was employed (method B).

Method B

Alternatively, the homogenized cell-free whole broth culture is passedthrough non-ionic polymeric resin (Supelco Diaion HP-20), washed withwater, and then methanol. The methanol eluate is further separated byreversed-phase HPLC (HP Zorbax Eclipse XDB-C8; 5 μm, 150×4.6 mm. Solventsystem: solvent A: acetonitrile/methanol/water 25:5:65, solvent B:acetonitrile/methanol/water 65:5:30. Gradient: start at 0 min withsolvent A and increase solvent B to 3% in 20 min. Flow=0.8 mL/min) toafford the same active peaks observed in method A (peaks A and B) andconfirmed by analytical HPLC using UV and MS detection. An HPLC trace isshown in FIG. 3.

Characteristics of Active Metabolites of NRRL No. B-30145

The impure fraction obtained from method A provided some initialinformation about the nature of the active metabolite. LC MS indicated amolecular weight [M+H⁺]=892.6 and the UV spectrum displays a shoulder at215-220 nm. 1D and 2D NMR suggests at least 2 propargyl alcohol segments[C═C—CH(OH)], several oxygenated methine carbons (X—CH—Y), and apossible sugar moiety. ¹H and ¹³C NMR are shown in FIGS. 4 and 5respectively.

Even though method B has not provided sufficient quantities for NMRanalysis, this method yielded cleaner peaks in sufficient amounts foranalysis by HPLC (octyl bonded silica gel) using UV and MS detectionmethods. Two major peaks (peak A and B) were obtained that matched thesame compounds identified as the active metabolites using method A (seeFIG. 3). The UV spectra of all compounds presented a shoulder at 215-220nm. LC MS of peak A indicated the presence of at least three (3)compounds with the following molecular weights [M+H⁺]=866.5, 882.5, and898.4 (see FIG. 6). Similarly, peak B showed at least three (3)compounds with molecular weights [M+H⁺]=892.5, 908.5, and 924.5 (seeFIG. 7).

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be apparent to those skilled in the art thatcertain changes and modifications will be practiced. Therefore, thedescription and examples should not be construed as limiting the scopeof the invention, which is delineated by the appended claims.

REFERENCES

-   Akasaki et al., “Monazomycin, a new antibiotic produced by a    streptomyces,” J. Antibiotics, vol. 16, pp 127-131 (1963).-   Axelrood et al., “Douglas-fir root-associated microorganism with    inhibitory activity towards fungal plant pathogens and human    bacterial pathogens,” Can. J. Microbiol., vol. 42, pp. 690-700    (1996).-   Campos et al., “[Griseocarnin, an antifungal antibiotic isolated    from Streptoverticillium griseocarneum (IA-7527)],” Rev Inst    Antibiot (Recife), vol. 14(1-2), pp. 91-100 (1974) (English Summary    only).-   Chamberlain and Crawford, “In vitro and in vivo antagonism of    pathogenic turfgrass fungi by Streptomyces hygroscopicus strains    YCED9 and WYE53, ” J of Industrial Microbiol Biotechnol, vol. 23,    pp. 641-646 (1999).-   Claridge et al., “New mitomycin analogs produced by directed    biosynthesis,” J. Antibiotics vol. 39, pp. 437-446.-   Crawford, “Use of Streptomyces bacteria to control plant pathogens,”    U.S. Pat. No. 5,527,526.-   Komori and Morimoto, “Isolation of the aromatic heptaenic    antibiotics trichomycin A-F by high-performance liquid    chromatography,” J. Chromatogr, vol. 481, pp 416 (1989).-   Lechevalier, H. A., and S. A. Waksman, “The actinomycetes.III    Antibiotics of actinomycetes”, The Williams & Wilkins Co.,    Baltimore, p430ff, (1962).-   Lechevalier, M. P, “Actinomycetes in agriculture and forestry,”    In M. Goodfellow, S. T. Williams, and M. Mordarski (ed.),    Actinomycetes in biotechnology. Academic Press, Inc., New York,    p.327-358 (1988).-   Mitscher, L. A. and M. P. Kunstmann, “The structure of neutramycin,”    Experientia vol. 25, pp. 12-3 (1969).-   Reddi, G. S., and A. S. Rao, “Antagonism of soil actinomycetes to    some soil-borne plant pathogenic fungi,” Indian Phytopathol. Vol.    24, pp. 649-657 (1971).-   Rothrock, C. S., and D. Gottlieb, “Role of antibiosis in antagonism    of Streptomyces hygroscopicus var. geldanus to Rhizoctonia solani in    soil,” Can. J. Microbiol., vol. 30, pp. 1440-1447, (1984).-   Schwinn et al., “Advances In Plant Pathology: Phytophthora    infestans, The Cause Of Late Blight Of Potato,” (Academic Press, San    Diego, p. 244 (1991).-   Suh, “Antifungal biocontrol agents, a process for preparing and    treating the same,” International Patent Publication Number WO    98/35017.-   Yuan and Crawford, “Characterization of Streptoimyces lydicus    WYEC108 as a potential biocontrol agent against fungal root and seed    rots,” Appl. Env. Microbiol. vol. 61, pp. 3119-28 (1995).

1. A composition comprising at least one isolated metabolite produced bya biologically pure Streptomyces sp. strain selected from a Streptomycessp. strain having all the identifying characteristics of the straindeposited with NRRL with Accession No. B-30145; and mutants of thestrain deposited with NRRL with Accession No. B-30145, wherein themutants have all the identifying characteristics of NRRL No. B-30145;and wherein the metabolite exhibits activity against plant pathogenicfungi, shows UV absorption between about 215 nm and about 220 nm and isnot aromatic.
 2. The composition of claim 1, wherein the metabolite hasa molecular weight [M+H+] between about 865 Daltons and about 925Daltons.
 3. The composition of claim 2, wherein the metabolite has themolecular weight selected from the group consisting of about 866.5Daltons, about 882.5 Daltons, about 898.4 Daltons, about 892.5 Daltons,about 908.5 Daltons and about 924.5 Daltons.
 4. The composition of claim1, wherein the metabolite is heat and base stable, is acid labile andhas a molecular weight [M+H+] between about 865 Daltons and about 925Daltons.
 5. The composition of claim 4, wherein the metabolite has themolecular weight selected from the group consisting of about 866.5Daltons, about 882.5 Daltons, about 898.4 Daltons, about 892.5 Daltons,about 908.5 Daltons and about 924.5 Daltons.
 6. The composition of claim1, wherein the metabolite comprises one or more chemical moietiesselected from the group consisting of an oxygenated methine carbon and asugar moiety.
 7. The composition of claim 1 further comprising acarrier.
 8. The composition comprising of claim 1 comprising more thanone metabolite and a carrier.
 9. The composition of claim 7, furthercomprising at least one chemical or biological pesticide.
 10. Thecomposition of claim 8, further comprising at least one chemical orbiological pesticide.
 11. The composition of any one of claims 7-10,wherein the composition is formulated as a formulation selected from thegroup consisting of a wettable powder formulation, a granuleformulation, an aqueous suspension, an emulsifiable concentrate, and amicroencapsulated formulation.
 12. An antifungal composition comprisingthe composition of claim 1 wherein the metabolite is isolated accordingto a method comprising: (a) loading a whole broth culture ofStreptomyces sp. strain NRRL No. B-30145 or mutants thereof that haveall the identifying characteristics of NRRL No. B-30145 onto a non-ionicabsorbent polymeric resin; (b) eluting the metabolite with an alcohol;(c) screening the eluent of step (b) with a bioassay for fractions ofthe eluent exhibiting antifungal activity; (d) loading the fractions ofthe eluent exhibiting antifungal activity of step (c) on a HPLC column;and (e) eluting the metabolite with an organic solvent, to produce theantifungal composition.
 13. The composition of claim 12, wherein theeluent of step (b) is methanol or a gradient of aqueous methanol. 14.The antifungal composition of claim 12, wherein the bioassay of step (c)is selected from the group consisting of the agar diffusion assay orslide germination assay.
 15. The antifungal composition of claim 12,wherein the organic solvent of step (e) is an acetonitrile-watergradient.
 16. A method for protecting or treating plants, fruit androots from a fungal infection comprising applying an effective amount ofthe composition of claim 1 to the plant, fruit or root.
 17. The methodof claim 16, wherein the fungal infection is caused by a fungus selectedfrom the group consisting of Alternaria solani, Botrytis cinerea,Rhizoctonia sp., Sclerotinia sp. and Phytophthora sp.
 18. The method ofclaim 16, wherein the composition contains more than one metabolite. 19.The method of claim 16, wherein the metabolite of the composition has amolecular weight [M+H+] between about 865 Daltons and about 925 Daltons.20. The method of claim 19, wherein the molecular weight of themetabolite is selected from the group consisting of about 866.5 Daltons,about 882.5 Daltons, about 898.4 Daltons, about 892.5 Daltons, about908.5 Daltons and about 924.5 Daltons.
 21. The method of claim 16,wherein the formulation comprises more than one metabolite.
 22. Themethod of claim 16, wherein the metabolite of the composition is heatand base stable, is acid labile and has a molecular weight [M+H+]between about 865 Daltons and about 925 Daltons.
 23. The method of claim22, wherein the metabolite has the molecular weight selected from thegroup consisting of about 866.5 Daltons, about 882.5 Daltons, about898.4 Daltons, about 892.5 Daltons, about 908.5 Daltons and about 924.5Daltons.
 24. The method of claim 16, wherein the composition is appliedas a formulation selected from the group consisting of a wettable powderformulation, a granule formulation, an aqueous suspension, anemulsifiable concentrate and a microencapsulation formulation.
 25. Themethod of claim 24, further comprising applying an effective amount ofat least one chemical or biological pesticide.